SOST Inhibits Prostate Cancer Invasion

Abstract

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

Fig 1. PC cell invasion.

A, schematic representation of the invasion assay. B, invasive potential of PC3 cells following 48 hours of co-culture with recombinant proteins. C, the effect of rWNT3a, rDKK1, and rSOST on the invasion of PC3, DU145, and C4-2Bm. D-E, dose response curve of PC3 cells to rSOST or rDKK1. F-G, Dose response curves of PC3 cell invasion when either rDKK1 or rSOST was held constant and the other was incrementally increased. H-K, representative images of PC3-mKate cells following co-culture conditions. Results are expressed as fold change ± SEM. * P<0.5, ** P<0.1, *** P<0.01.

Fig 2. PC cell invasion towards osteoblasts isolated from neo-natal mice calvaria.

A, schematic representation showing OBs grown on the bottom of the chamber. B, invasive potential of PC3 cells towards control OBs, OBs with elevated Wnt signaling, and OBs lacking Wnt signaling. C-F, representative images of PC3-mKate cells following co-culture conditions. G-J, invasive potential of PCa cells with different osteolytic potential towards each OB. Results are expressed as fold change ± SEM. * P<0.5, ** P<0.1, *** P<0.01.

Fig 3. Downstream Wnt activity following 48hrs of co-culture.

A, TopFlash reporter Luciferase assay in PC3 cells co-cultured with OBs. B, TopFlash reporter assay in PC3 cells co-cultured with DKK1. C-J, activated β-catenin (ABC) immunohistochemistry in PC3 cells under multiple conditions; green (ABC), blue (DAPI). Results are expressed as fold change ± SEM. * P<0.5, ** P<0.1, *** P<0.01.

Fig 4. Molecular changes in invasive PC3 cells.

A, microarray analyses of PC3 cells co-cultured with OB^SostKO, OB^WT+ rhDKK1, and OB^WT + rhSOST as compared to monocultures; overlay representation of upregulated (green) and downregulated (red) genes. B, representative list of upregulated transcripts. C, representative images of CRIM1 protein expression modulation by rhDKK1 and rhSOST; green (CRIM1), blue (DAPI). Results are expressed as fold change ± SEM. * P<0.5, ** P<0.1, *** P<0.01. D, PC3 transfected with a CMV-Crim1 expression vector were significantly more invasive then PC3 cells (p-value 0.006).

Fig 5. Morphological changes in PC3 cells.

A-C, Representative SEM images (1500x) of PC3 cells treated with rhDKK1 (B, b), or rhSOST (C, c). D-H, Immunofluorescence staining of PC3 cells treated with rhDKK1 (E), rhSOST (F), rhCRIM1 (G) and rhWNT3A (H); anti–CRIM1 (green) and rhodamine-conjugated phalloidin (blue).

Fig 6. SOST reduces PC3-mediated osteolysis in xenograft derived tumor lesions.

Representative femur micro-CT scans from PC3 (A), PC3 ^DKK1 (B) and PC3 ^SOST (C) injected NSG mice (N = 6/group). Bone volume was quantified for both PC injected and uninjected contralateral femurs, and relative bone loss due to osteolysis was calculated for each group by subtracting the injected (L) from the uninjected (R) values (D). PC ^SOST injected femurs experienced significantly less bone loss due to advanced osteolytic lesions (*p<0.05).